Contact with antigen-presenting cells initiates an activation cascade within T lymphocytes, including a rise in cytosolic calcium, lymphokine production, and cell division. Calcium imaging combined with an optical trap enabled the T-cell contact requirements and polarity to be investigated at the single-cell level. Anti-CD3 mAb-coated beads induced calcium signaling with ~ ten-fold higher frequency upon contact with the leading edge of the T cell, compared with the trailing edge. Engagement of at least 340 T cell receptors (~1% of the total on the cell) was required to initiate Ca2+ signaling, and the minimal contact area was ~3 (m2. Our results indicate that ~1000 TCRs are required to generate a long-lasting [Ca2+]i signal which might be required for gene expression. We expect this approach can be combined with ligands for accessory molecules or with downstream assays for gene expression in order to examine species and clonal-specific variations in the T cell resp onse and deepen our understanding of the relationship between early activation events and gene expression.